A multifunctional cross-validation high-throughput screening protocol enabling the discovery of new SHP2 inhibitors

Abstract

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC 50 = 0.32 μmol/L; WS-635: IC 50 = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC 50 = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins.

Harnessing Reversed Allosteric Communication: A Novel Strategy for Allosteric Drug Discovery

“…Overall, the emergence of a reversed allosteric communication strategy, combined with a comprehensive allosteric database (available online at ) and advanced structural prediction approaches, is expected to accelerate the end-to-end methodology trend of allosteric drug design. − The reversed allosteric communication strategy opens up a formidable methodology for allosteric drug design and drug resistance treatment and further implies the mystical regulation of multisite communication. In conclusion, the reversed allosteric communication theory provides a novel strategy for both academia and industry to identify potential allosteric sites for the discovery of allosteric modulators.…”

SHP2 allosteric inhibitor TK-453 alleviates psoriasis-like skin inflammation in mice via inhibition of IL-23/Th17 axis

“…Since the first allosteric inhibitor SHP099 was reported in 2016, several effective SHP2 inhibitors have been identified ( Chen et al., 2016 ). The allosteric mechanism “molecular glue” targeting SHP2 inhibitors has aroused special interest in this long-sought goal ( LaMarche et al., 2020 ; Nichols et al., 2018 ; Song et al., 2021a ; Xie et al., 2017 ). (3) PROTAC degrader of SHP2 protein.…”

Crystallographic landscape of SHP2 provides molecular insights for SHP2 targeted drug discovery

“…Src homology‐2‐containing protein tyrosine phosphatase 2 (SHP2), a member of protein tyrosine phosphatases (PTPs) family, is a dephosphorylase encoded by PTPN11 . Located at downstream of several receptor tyrosine kinases (RTKs) in the membrane, SHP2 acts as a convergent node to provide crosstalk for several signaling pathways, including PI3K‐AKT‐mTOR, Ras‐Raf‐MEK‐ERK, JAK‐STAT, and PD‐1/PD L‐1 pathways 1 . Under physiological or pathogenic conditions, SHP2 undergoes dynamic transition between closed and open conformations, causing structural reorganization and phosphatase activity alternation with different degrees 2 .…”